proteomic analysis of marinobacter hydrocarbonoclasticus
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Proteomic analysis of Marinobacterhydrocarbonoclasticus SP17 biofilm formation at the
alkane-water interface reveals novel proteins and cellularprocesses involved in hexadecane assimilation
Pierre-Joseph Vaysse, Laure Prat, Sophie Mangenot, Stéphane Cruveiller,Philippe Goulas, R. Grimaud
To cite this version:Pierre-Joseph Vaysse, Laure Prat, Sophie Mangenot, Stéphane Cruveiller, Philippe Goulas, et al..Proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm formation at the alkane-waterinterface reveals novel proteins and cellular processes involved in hexadecane assimilation. Researchin Microbiology, Elsevier, 2009, 160 (10), pp.829-837. �10.1016/j.resmic.2009.09.010�. �hal-01614789�
1
Proteomic analysis of Marinobacter hydrocarbonoclasticus SP17 biofilm formation at the 1
alkane-water interface reveals novel proteins and cellular processes involved in 2
hexadecane assimilation. 3
4
Pierre-Joseph Vayssea, Laure Prata†, Sophie Mangenotb, Stéphane Cruveillerc, Philippe 5
Goulasa, Régis Grimauda* 6
7
a Institut Pluridisciplinaire de Recherche en Environnement et Matériaux, Equipe 8
Environnement et Microbiologie UMR5254 CNRS, IBEAS, Université de Pau et des Pays de 9
l’Adour, BP1155, 64013 Pau cedex, France 10
11
b CEA/DSV/IG/Genoscope, 2 rue Gaston Cremieux, 91057 Evry cedex, France 12
13
c CEA/DSV/IG/Genoscope/LGC, 2 rue Gaston Cremieux, 91057 Evry cedex, France 14
15
† Current address : EPFL, ENAC-ISTE, Laboratory of Environmental Biotechnology, 16
Lausanne, Switzerland 17
18
E-mail addresses of all authors 19
• Pierre-Joseph Vaysse: [email protected] 20
• Laure Prat: [email protected] 21
• Sophie Mangenot: [email protected] 22
• Stéphane Cruveiller: [email protected] 23
• Philippe Goulas: [email protected] 24
• Régis Grimaud: [email protected] * Correspondence and reprints 25
Tel: 33 (0)5 59 40 74 86, Fax: 33 (0)5 59 40 74 94 26
27
2
Abstract 28
Many hydrocarbon degrading bacteria form biofilms at the hydrocarbon-water interface to 29
overcome the low accessibility of these poorly water-soluble substrates. In order to gain 30
insight into the cellular functions involved, we undertook a proteomic analysis of 31
Marinobacter hydrocarbonoclasticus SP17 biofilm developing at the hexadecane-water 32
interface. Biofilm formation on hexadecane led to a global change of the cell physiology 33
involving modulation of the expression of 573 out of 1144 detected proteins when compared 34
with planktonic cells growing on acetate. Biofilm cells overproduced a protein, encoded by 35
MARHY0478 that contains a conserved domain belonging to the family of the outer 36
membrane transporters of hydrophobic compounds. Homologs of MARHY0478 were 37
exclusively found in marine bacteria degrading alkanes or possessing alkane degradation 38
genes and hence presumably constitute a family of alkane transporter specific to marine 39
bacteria. Interestingly, we also found that sessile cells growing on hexadecane overexpressed 40
type VI secretion system components. This secretion system has been identified as a key 41
factor in virulence and in symbiotic interaction with host organisms. This observation is the 42
first experimental evidence of the contribution of a type VI secretion system to environmental 43
adaptation and raises the intriguing question about the role of this secretion machine in alkane 44
assimilation. 45
46
Keywords: Marinobacter hydrocarbonoclasticus SP17; biofilm; alkane degradation; 47
proteomic; type VI secretion system 48
3
49
1. Introduction 50
51
Biofilm formation at the hydrocarbon-water interface has been observed with various 52
alkane degrading strains, e.g. Rhodococcus sp. Q15 [34], Acinetobacter venetianus RAG-1 [2] 53
and Oleiphilus messinensis [9] as well as polycyclic aromatic hydrocarbons (PAHs) 54
degrading strains, e.g. Pseudomonas sp. strain 8909N [19], Sphingomonas sp.CHY-1 [35] and 55
Mycobacterium frederiksbergense LB501T [3]. 56
The ecological significance of these biofims has been demonstrated by a study devoted to the 57
diversity of biofilm communities developing on PAHs [27]. It has been shown that biofilm 58
communities contained a greater diversity of active species and of PAH degradation genes 59
than the planktonic communities enrichment. Furthermore the diversity of active species 60
found in the biofilm closely matched that of the PAH-contaminated soil used as inoculum. 61
These findings suggest that biofilm formation on hydrocarbons is a probable lifestyle in 62
natural ecosystems. 63
Biofilms developing on hydrocarbons present two properties distinguishing them from 64
other biofilms which are their substrate-substratum specificity and their capacity to overcome 65
the low accessibility of hydrophobic substrates. It was observed in the case of PAH-degrading 66
strains that the less soluble the hydrocarbon was, the more cells grew at the PAH-water 67
interface [12,23]. Biofilm formation has been shown to promote growth on hydrocarbons by 68
facilitating interfacial access. Kinetic studies demonstrated that growth at the interface occurs 69
faster than mass transfer rate of hydrocarbons in the absence of bacteria would suggest 70
[4,5,10,13]. Thus, biofilm formation constitutes an efficient adaptive strategy to assimilate 71
hydrocarbon. However, the genetic determinants and the molecular mechanisms underlying 72
the functioning of these biofilms remain poorly understood. In order to identify the proteins 73
4
and cellular functions involved, we undertook a proteomic analysis of the biofilm of 74
Marinobacter hydrocarbonoclasticus SP17 growing at the hexadecane-water interface. This 75
marine alkane degrading bacterium readily forms biofilms on metabolizable hydrophobic 76
organic compounds including n-alkanes from 8 to 28 carbon atoms, whereas in the same 77
condition this strain does not develop biofilms on an inert substratum such as plastic or glass 78
[15]. The doubling time of biofilm cells growing on hexadecane (between 3 and 5 hours 79
depending on the development stage of the biofilm) is similar to that observed on water-80
soluble substrates such as acetate, pyruvate and lactate (P-J Vaysse, unpublished results). 81
Furthermore, the rate of hexadecane degradation dramatically decreases when the biofilm is 82
disorganized by strong shaking, indicating a strong link between biofilm formation and the 83
utilization of alkanes [15]. Theses observations indicate that the biofilm lifestyle must provide 84
M. hydrocarbonoclasticus SP17 with efficient mechanisms to access the hexadecane. In the 85
current study, we undertook a proteomic analysis of a biofilm growing at hexadecane-water 86
interface. The results obtained indicate that adaptation to alkane utilization as carbon and 87
energy source involves a global change in cell physiology. Novel proteins and cellular 88
processes involved in alkane assimilation were revealed. 89
5
2. Material and methods 90
91
2.1 Growth conditions and preparation of protein extracts 92
93
The bacterial strain used in this study was Marinobacter hydrocarbonoclasticus SP17 94
(ATCC 49840). Cultures were carried out in synthetic sea water (SSW) [8]. 95
All chemicals used were from Sigma-Aldrich unless otherwise specified. Biofilm cultures 96
were inoculated with cells growing exponentially on acetate (20 mM) washed twice with one 97
volume of SSW and resuspended to a final optical density at 600nm of 0.1 in 300 ml SSW 98
medium supplemented with 0.2% (v/v) hexadecane in 1 l Erlenmeyer flasks. Biofilms were 99
grown at the hexadecane-SSW interface at 30 °C, under slow shaking (50 rpm). After 35 100
hours incubation, the medium was carefully discarded, the biofilms resuspended in 30 ml of 101
SSW and centrifuged for 20 min at 20,000 g at room temperature. Biofilms were harvested 102
above the supernatant while residual planktonic cells were pelleted. Cells grown on acetate 103
(100 ml) were harvested during exponential growth phase (OD600nm= 0.3) by centrifuging 104
20 min at 20,000 g at room temperature. 105
For protein extraction, cell pellets or biofilms were washed twice with 5 ml of acetone, 106
resuspended in 5 ml of water containing a protease inhibitor cocktail and sonicated on ice for 107
1 min with 500 ms/s pulses at 35 W. Sonicated cell suspensions were then incubated with 40 108
µg of DNase I, 10 µg of RNase A plus 0.01% (v/v) Triton X100 for 30 min at room 109
temperature. Proteins were precipitated for 30 min at 4 °C by adding 500 µl of 100% 110
trichloroacetic acid and then centrifuged 10 min at 20,000g at 4 °C. Protein extracts were 111
washed twice with 1 ml of trichloroacetic acid 10% (v/v) and twice with 1 ml of acetone. 112
Proteins were air dried and dissolved in IEF buffer (urea 8 M, 3-[(3-113
Cholamidopropyl)dimethylammonio]-1-propanesulfonate 4% (w/v), dithiothreitol 60 mM, 114
6
Pharmalyte 3-10 2% (v/v) (Amersham Biosciences) and bromophenol blue 0.0002% (w/v)). 115
Protein concentration was estimated using Biorad Protein assay and finally adjusted to 1 µg/µl 116
with IEF buffer. 117
118
2.2 2D electrophoresis and gel analysis 119
120
Two hundred and thirty micrograms of protein were applied to a 24 cm Immobiline 121
Dry strip with a 3-7 non linear pH gradient (GE Healthcare). Isoelectric focusing and SDS-122
PAGE (12.5%) were carried out using the Multiphor II and Ettan DALTsix systems, 123
respectively (GE Healthcare) according to the manufacturer’s instructions. Gels were stained 124
with Deep Purple Total Protein Stain (GE Healthcare) and then scanned using a Typhoon 125
9200 fluorescent scanner (GE Healthcare). Image analysis, spot detection and matching were 126
carried out using Image Master Platinum software (GE Healthcare) and checked manually. 127
Three replicate gels from three independent experiments were run for each growth condition. 128
The normalized protein amount for each protein spot was defined as the fraction of that spot 129
volume to the total spot’s volume of the gel. Student t-test (P<0.05) and a threshold of 2-fold 130
change were used to determine the proteins significantly differentially expressed between the 131
two conditions. 132
133
2.3 Protein identification by nanoLC-MS/MS 134
135
Protein spots were excised from gels, destained in ammonium bicarbonate 50 mM 136
50% acetonitrile (ACN), rinsed twice in ultrapure water and dehydrated in 100% ACN. After 137
ACN removal by evaporation, gel pieces were dried, rehydrated with a trypsin solution (10 138
ng/ml in 50 mM ammonium bicarbonate) at 4 °C for 10 min and finally incubated overnight 139
7
at 37 °C. The supernatant was collected by two successive extractions with 140
H2O/ACN/HCOOH (47.5/47.5/5), pooled, and concentrated in a vacuum centrifuge to a final 141
volume of 25 µl. The peptide mixture was analyzed by on-line capillary HPLC (LC Packings, 142
Amsterdam, The Netherlands) coupled to a nanospray LCQ Deca XP Ion Trap mass 143
spectrometer (Thermo-Finnigan, San Jose, CA). MS/MS data were acquired using a 2 m/z 144
units ion isolation window and a 35% relative collision energy. Data were searched using 145
SEQUEST through Bioworks 3.3.1 interface (ThermoFinnigan) against the M. 146
hydrocarbonoclasticus SP17 whole genome sequence. Gene numbers, gene function, and 147
functional category are presented according to the unpublished annotated genome. 148
149
2.4 Database searching and sequence analysis 150
151
Sequences similarities were searched against the translation of the non-redundant 152
GenBank CDS database, using the BLAST program [1]. Homologs were defined as proteins 153
giving an alignment with a bits score above 100 (scoring matrix BLOSUM62) and an 154
expected value (E-value) below 1.10-25. Protein domain searches and multiple sequence 155
alignment were carried out using the NCBI-Conserved Domain Database search program with 156
CDD v2.16-27036 PSSMs database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) 157
[18]. Synteny similarities were searched using the MAGE microbial genome annotation 158
system from the Genoscope Evry France 159
(https://www.genoscope.cns.fr/agc/mage/wwwpkgdb/MageHome). 160
8
161
3. Results and discussion 162
163
3.1. Comparison of protein patterns of biofilm cells growing on hexadecane with planktonic 164
cells growing on acetate 165
166
In order to characterize the molecular mechanisms involved in the development of 167
biofilm on alkanes, we compared the proteomes of biofilm growing on hexadecane and 168
planktonic cells growing on acetate, hereafter referred to as BH and PA respectively (Fig. 1). 169
A total of 1144 spots appearing on all three replicates of at least one growth condition were 170
detected. 576 spots (50%) were found to change significantly between PA and BH. Among 171
these proteins, 245 were overexpressed in BH and 331 in PA. 81% of the differentially 172
expressed protein had an induction ratio above 10. This high proportion of differentially 173
expressed proteins is unusual when compared to other similar proteomic analyses. For 174
instance, a study of Alcanivorax burkomensis SK2 comparing growth on hexadecane with 175
growth on pyruvate revealed 97 proteins differentially expressed [24]. However, A. 176
burkomensis SK2 protein extracts were prepared from whole cultures consisting of a mixture 177
of biofilm cells and detached planktonic cells. Therefore, the expression protein profiles 178
might reflect the average protein expression levels from different cellular states, thus 179
lessening the actual expression fold-change of one specific condition. Furthermore, in our 180
study, proteins were revealed using a fluorescent dye with a higher sensitivity and larger 181
dynamic range than Coomassie blue. 182
The high number of differentially expressed proteins between the biofilm cells 183
growing on hexadecane and those growing on acetate signify that growth on hexadecane 184
involve a global change in the cell physiology, requiring numerous cellular functions. This 185
9
could be explained by the change of two major factors between the compared conditions: the 186
lifestyle (biofilm or planktonic) and the carbon source (hexadecane or acetate). In many 187
species, biofilm formation has been shown to require a large number of protein functions. The 188
biofilm lifestyle induces changes in the environmental conditions encountered by cells, such 189
as the formation of nutrient microgradients. The adaptation to these new conditions requires 190
most likely a great number of proteins [28]. On the other hand, the response to alkanes 191
involves the modulation of the expression of numerous proteins required for the transport, the 192
metabolism or to avoid toxic effects of these compounds [31]. A total of 58 spots, that could 193
unambiguously be assigned to a single protein, were kept for further analysis (Table 1). The 194
major type IV pilus subunit, PilA (encoded by MARHY2564) was under-produced in BH. 195
Type IV pili have been shown to be involved in host cell adhesion, biofilm formation, DNA 196
uptake and twitching motility [21]. A DNA microarray analysis showed that pilin genes were 197
repressed in the biofilm of Pseudomonas aeruginosa [33]. Furthermore, the type IV pilus of 198
P. aeruginosa has been shown to have multiple effects on biofilm formation mainly through 199
twitching motility and adhesion [11,20]. 200
Proteins involved in nutrient transport across cellular envelopes constitute a large class 201
of proteins overproduced in BH. This includes components of phosphate (encoded by 202
MARHY3535 pstS) and thiosulfate (encoded by MARHY2019 cysP) ABC transporters. Iron 203
uptake and transport proteins were represented by FhuE, FbpA and CirA encoded by 204
MARHY1035, MARHY2192 and MARHY3135 respectively. Four other proteins coding for 205
putative ABC transporters and porins (encoded by MARHY0256, MARHY0299, 206
MARHY3277, MARHY34332 and MARHY3166) were also found, although their actual 207
function remain uncertain. Increased capacities in solute transport may reflect an adaptation to 208
the constraints imposed by the biofilm lifestyle. Indeed, the increase in biofilm thickness is 209
thought to hinder nutrient penetration into the deepest layers of the biofilm. Thus, cells would 210
10
require an increased capacity in nutrient uptake and transport. This is particularly important in 211
seawater where iron and phosphate are in limiting concentrations. Micronutrients such as iron 212
and inorganic phosphate are also known to strongly influence biofilm development by acting 213
as environmental cues regulating biofilm formation [25]. 214
215
3.2. Redirection of carbon flux in biofilm cells growing on hexadecane 216
Most of the enzymes corresponding to the CO2-releasing steps of the tricarboxylic acid 217
cycle (TCA cycle) (encoded by MARHY0078 idh, MARHY2120 sucD, MARHY2121 sucC, 218
MARHY2126 sdhA) were found to be down regulated in BH. In addition, the gene encoding 219
malate synthase (MARHY1458 glcB), catalyzing the conversion of glyoxylate into malate, 220
was upregulated. This modulation of TCA cycle enzymes suggests a stimulation of the 221
glyoxylate bypass in BH. This anaplerotic pathway allows the replenishment of cells in the 222
metabolic intermediates necessary for the synthesis of their cellular components when acetyl-223
CoA is the only carbon source available in the cell [16]. The activation of the glyoxylate 224
bypass under a hexadecane diet compared to a diet of pyruvate or glucose has been already 225
observed in proteomic analyses on A. burkomensis SK2 and Geobacillus thermodenitrificans 226
[6,24]. In our study, stimulation of the glyoxylate bypass was observed under hexadecane 227
conditions compared with acetate. In hexadecane and acetate conditions, the intracellular 228
carbon source is acetyl-CoA hence the glyoxylate pathway is required in both conditions. The 229
explanation of the glyoxylate bypass stimulation could lie in the fact that hexadecane is a 230
more energetic substrate than acetate. In fact the breakdown of hexadecane to acetyl-CoA 231
produces energy whereas the conversion of acetate to acetyl-CoA does not. Augmenting the 232
flux of carbon through the glyoxylate bypass on hexadecane could allow cells to restore the 233
balance between carbon assimilation and energy production. However interpretation of 234
11
proteomic data with regard to metabolic flux should be taken with caution, since metabolic 235
pathways can be regulated at the level of enzyme activity, in addition to gene expression. 236
Fatty acid biosynthesis genes fabA, fabB and fabF (MARHY3086, MARHY3087 and 237
MARHY1438, respectively) were found to be down regulated in BH. This corroborates the 238
fact that in cells growing on hexadecane the main fatty acids of cellular lipids were derived 239
from the oxidation of alkanes [26]. 240
241
3.3. A type VI secretion system is overproduced by biofilm cells growing on hexadecane 242
243
Three proteins sharing similarities with type VI secretion system (T6SS) subunits, 244
encoded by MARHY3623, MARHY3634 and MARHY3635, were overexpressed in BH. 245
MARHY3634 and MARHY3635 were among the most abundant protein detected in BH, with 246
0.2 and 0.7 % of total protein respectively. MARHY3634, MARHY3635 and MARHY3623 247
are localized within a cluster of 16 genes (from MARHY3635 to MARHY3620) that are 248
transcribed in the same direction. All members of the M. hydrocarbonoclasticus SP17 cluster 249
share similarity with genes found in T6SS clusters from other bacteria including Vibrio 250
cholerae, P. aeruginosa and Escherichia coli. The best synteny conservation with a 251
characterized T6SS was observed with Vibrio cholerae N16961 T6SS gene cluster (Fig. 2). 252
Homologs of component of T6SS already characterized, i.e. Dot, IcmF, and ClpV (a 253
subfamily of ClpB ATPase), were also found in M. hydrocarbonoclasticus SP17 cluster 254
(MARHY3626, MARHY3621 and MARHY3625). Vgr and Hcp are two proteins secreted 255
through T6SS. One homolog of Vgr is found (MARH3620) within this cluster while other 256
homologs of Vgr and Hcp are found elsewhere on the M. hydrocarbonoclasticus SP17 257
chromosome [7] (Fig. 3). Experimental evidence of the functionality of homologs of 258
MARHY3635 and 3634 was provided by studies of the pathogen Edwardsielle tarda [22]. 259
12
Deletion mutants of evpA and evpB (MARHY3635 and MARHY3634, respectively) were 260
impaired in protein secretion and showed reduced virulence in blue gourami fish. Based on 261
the high degree of sequence similarity and synteny conservation between characterized T6SS 262
clusters, we propose that the genes from MARHY3635 to MARHY3620 constitute a T6SS 263
gene cluster. The functionality of this T6SS is supported by the detection of three proteins 264
encoded by this cluster. 265
Although type VI secretion systems have been identified as key factors in virulence of 266
pathogenic bacteria and in symbiotic interaction with host organisms, in silico analyses have 267
revealed their presence in many species that are not considered as pathogens or symbionts. 268
This led to the hypothesis that T6SS may also contribute to environmental adaptation [29]. 269
Our data provide the first experimental evidence of the production of a T6SS during biofilm 270
development by an environmental bacterium. The role of T6SS in biofilm formation during 271
growth on hexadecane by M. hydrocarbonoclasticus SP17 remains puzzling. T6SS may be 272
required to secrete proteins needed for biofilm formation or for efficient assimilation of 273
carbon source difficult to access. 274
275
3.4. Evidence for an alkane transporter family specific to marine bacteria 276
277
Two proteins produced in large quantities in BH, encoded by MARHY0478 and 278
MARHY0477, drew our attention because their sequences were found to be conserved 279
exclusively in marine species capable to use alkanes as a carbon source or possessing at least 280
one copy of an alkane hydroxylase gene. The phylogenetic distribution of MARHY0478 and 281
MARHY0477 homologs is rather restricted since they are found only in two orders of the 282
Gammaproteobacteria: the Oceanospirillales and the Alteromonadales. The degree of peptide 283
sequence identity varies between 29% and 98%. Interestingly, these proteins are found in 284
13
Alcanivorax borkumensis SK2, a key alkane degrader in polluted seawater. Several strains 285
possess multiple homologs of MARHY0478 and MARHY0477 with a maximum of six in 286
Alcanivorax sp DG881 (Table 2). 287
In M. hydrocarbonoclasticus SP17, MARHY0478 and MARHY0477 are separated by only 288
11 base pairs and are transcribed in the same direction. This spatial organization is indicative 289
of a putative operon. Such an operon would suggest that the products of these genes are 290
involved in the same function. An operon structure is likely to be conserved in M. 291
hydrocarbonoclasticus VT8 and A. burkumensis SK2 since these strains contain homologs of 292
MARHY0477 and MARHY0478 that are adjacent and transcribed in the same direction. 293
The strong expression of MARHY0478 and MARHY0477 in biofilm on hexadecane indicates 294
that they might play an important role in alkane assimilation. In order to gain more insight 295
into their function, conserved domains in the two proteins were searched against the NCBI 296
Conserved Domain Database using RPS-BLAST [18]. Only the protein encoded by 297
MARHY0478 gave a positive hit (E-value = 10-13) with the pfam03349 domain family. This 298
conserved domain is found in outer membrane proteins transporting hydrophobic compounds 299
out of Gram negative bacteria. This family includes the monoaromatic hydrocarbon transport 300
proteins TodX from Pseudomonas putida F1 and TbuX from Ralstonia pickettii PKO1, and 301
the long chain fatty acid tranporter FadL from Escherichia coli [14,30,32]. Fig. 3 shows the 302
multiple peptide sequence alignment for the conserved domain including the amino acid 303
sequence deduced from MARHY0478, TodX, TbuX and FadL. This alignment is based upon 304
the frequency of each amino acid substitution in the set of protein used to create the conserved 305
domain model [17]. Peptide encoded by MARHY0478 exhibited a clear sequence similarity 306
with the four hydrophobic compounds transporters. In view of this significant domain 307
conservation, it is very likely that MARHY0478 encodes for a hydrophobic compound 308
transporter. Given that MARHY0478 was induced during growth on hexadecane, and that 309
14
homologs are only found in marine alkane degrading strains or putative alkane degrading 310
strains, we hypothesized that MARHY0478 and its homologs would constitute a family of 311
alkane transporter specific to marine bacteria. The production in large quantity of an alkane 312
transporter in biofilm cells would contribute to the high growth rate observed on alkane 313
despite its low solubility. 314
Polypeptides encoded by MARHY0477 and their homologs define a protein family 315
whose function is likely to be related to MARHY0478, as members of the two families co-316
occur in the same strains and occasionally form a putative operon. The ecological significance 317
of the MARHY0478 and MARHY0477 families certainly deserve closer inspection as they 318
are present in genera known to be the main actors in hydrocarbon degradation in marine 319
environments. 320
321
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322
Acknowledgments 323
We thank Dr Rizard Lobinsky, Dr Hugues Prudhomme and the Pôle Protéomique of 324
the Bordeaux 2 University for protein identification. We also thank Dr Pierre Sivadon and Dr 325
Anne Fahy for useful discussions and critical reading of the manuscript. We gratefully 326
acknowledge the 6th European Framework Programme, Contract 018391 FACEIT, the 327
National Program ANR “ECCO” INDHYC project, the CNRS program Ingénierie 328
Ecologique, the région Aquitaine and the département des Pyrénées Atlantiques for financial 329
support. 330
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430
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Legends to figures 431
432
Fig. 1. Two-dimensional gel electrophoresis of protein extracts of M. hydrocarbonoclasticus 433
SP17. Planktonic cells grown on acetate (top image) and biofilm cells grown on hexadecane 434
(bottom image). Spots of identified proteins are numbered. Molecular weights (in kDa) are 435
indicated on the left side. 436
437
Fig. 2. Organization of the genes encoding for type VI secretion system of M. 438
hydrocarbonoclasticus SP17 (top) and Vibrio cholerae N16961 (bottom). The values between 439
homologous genes refer to the percent peptide sequence identity of their products. Accession 440
number : MARHY3635 to MARHY3620 (FP475883 to FP47598), MARHY0176 441
(FP475953), MARHY1076 (FP475938) and MARHY2494 (FP475918) 442
443
Fig. 3. Multiple sequence alignment of FadL, TbuX, TodX and translated MARHY0478. 444
Sequence alignment was performed using the web-based tool CD search using the PSSM 445
112176 scoring matrix. Conserved residues are shown in grey boxes and identical residues in 446
black boxes. Secondary structures elements of FadL were retrieved from the PDB database 447
(PDB ID:1t16) and are shown above the sequences. 448
449
22
Tables Table 1
Identification of proteins differentially expressed in biofilm on hexadecane compared to planktonic cells on acetate.
Spot NO.
CDS Gene Product Differential abundancea
Amount in planktonic/acetate
conditionb
Amount in biofilm/hexadecane
conditionb
Gene accession numberc
Transport and binding protein
1 MARHY1035 fhuE Outer membrane receptor for ferric iron uptake +5.01 0.0102 0.0513 FP475940 2 MARHY2192 fbpA Iron(III) ABC transporter, periplasmic iron(III)-binding
protein +3.89 0.1233 0.4793 FP475923
3 MARHY3135 cirA Ferric iron-catecholate outer membrane transporter BH ND 0.079 FP475909 4 MARHY2019 cysP Thiosulfate transporter subunit +3.01 0.1170 0.3525 FP475928 5 MARHY3535 pstS ABC phosphate transporter, periplasmic component +10.74 0.1018 1.0934 FP475901 6 MARHY3277 _ ABC-type metal ion transporter, periplasmic component BH ND 0.0831 FP475905 7 MARHY1478 _ ABC-type branched-chain amino acid transporter, periplasmic
component -4.32 0.0882 0.0204 FP475934
Cellular processes
8 MARHY2564 pilA Fimbrial protein precursor -2.44 0.6983 0.2861 FP475917 9 MARHY2994 tpm Thiopurine methyltransferase PA 0.0551 ND FP475913
Information transfer 10 MARHY3200 greA Transcription elongation factor BH ND 0.0843 FP475907
Protein fate 11 MARHY0922 _ FKBP-type peptidyl-prolyl cis-trans isomerase PA 0.0418 ND FP475944 12 MARHY0942 dsbC Protein disulfide isomerase II +4.44 0.0363 0.1611 FP475943 13 MARHY0958 mucD Serine protease +4.05 0.0517 0.2091 FP475942 14 MARHY1798 slyD FKBP-type peptidyl-prolyl cis-trans isomerase PA 0.0584 ND FP475931
23
Spot NO.
CDS Gene Product Differential abundancea
Amount in planktonic/acetate
conditionb
Amount in biofilm/hexadecane
conditionb
Gene accession numberc
Stress response
15 MARHY3764 ahpF Alkyl hydroperoxide reductase f-subunit BH ND 0.0369 FP475881
Cofactor biosynthesis 16 MARHY2568 coaE Dephospho-CoA kinase PA 0.0533 ND FP475916
Aminoacid biosynthesis 17 MARHY1723 asd Aspartate-semialdehyde dehydrogenase PA 0.0655 ND FP475932 18 MARHY3112 hisH Glutamine amidotransferase BH ND 0.1733 FP475910 19 MARHY0406 lysA Diaminopimelate decarboxylase PA 0.0901 ND FP475949 20 MARHY2259 serC 3-phosphoserine/phosphohydroxythreonine aminotransferase BH ND 0.1327 FP475920
Nucleotide biosynthesis 21 MARHY2255 cmk Cytidylate kinase BH ND 0.0691 FP475921 22 MARHY2158 ndk Multifunctional nucleoside diphosphate kinase and
apyrimidinic endonuclease and 3'-phosphodiesterase BH ND 0.0836 FP475924
23 MARHY3664 pyrC Aspartate carbamoyltransferase, non-catalytic chain BH ND 0.0533 FP475882 24 MARHY2249 pyrF Orotidine-5'-phosphate decarboxylase +3.35 0.0582 0.1948 FP475922
Central intermediary metabolism 25 MARHY3262 exaC NAD+ dependent acetaldehyde dehydrogenase -3.44 0.0968 0.0281 FP475906
Energy metabolism 26 MARHY1444 acnB Bifunctional aconitate hydratase 2 and 2-methylisocitrate
dehydratase -2.89 0.9104 0.3147 FP475936
27 MARHY0078 idh Isocitrate dehydrogenase -3.13 0.2014 0.0643 FP475954 28 MARHY2120 sucD Succinyl-CoA synthetase, NAD(P)-binding, alpha-subunit -3.2 0.8657 0.2708 FP475927 29 MARHY2121 sucC Succinyl-CoA synthetase, beta-subunit -6.97 0.1894 0.0272 FP475926 30 MARHY2126 sdhA Succinate dehydrogenase, flavoprotein subunit -4.35 0.3639 0.0837 FP475925 31 MARHY0774 petA Ubiquinol-cytochrome c reductase iron-sulfur subunit +6 0.0449 0.2696 FP475945 32 MARHY1458 glcB Malate synthase G BH ND 0.0203 FP475935 33 MARHY1802 etfA Electron transfer flavoprotein subunit, FAD-binding +3.13 0.1707 0.5347 FP475930 34 MARHY1815 paaG Enoyl-CoA hydratase PA 0.073 ND FP475929
24
Spot NO.
CDS Gene Product Differential abundancea
Amount in planktonic/acetate
conditionb
Amount in biofilm/hexadecane
conditionb
Gene accession number
Fatty acid biosynthesis
35 MARHY1009 acs Acetyl-CoA synthetase PA 0.0435 ND FP475941 36 MARHY3086 fabA Beta-hydroxydecanoyl thioester dehydrase PA 0.1247 ND 37 MARHY3087 fabB 3-oxoacyl-[acyl-carrier-protein] synthase I -2.54 0.5215 0.2056 FP475911 38 MARHY1438 fabF 3-oxoacyl-[acyl-carrier-protein] synthase II -4.04 0.1566 0.0388 FP475937 39 MARHY1579 prpE Propionate-CoA ligase -5.75 0.1203 0.0209 FP475933
Uncertain or unknown function 40 MARHY0256 _ Probable ABC transporter, ATPase subunit PA 0.2227 ND FP475952 41 MARHY0299 _ PropableTRAP dicarboxylate transporter BH ND 0.123 FP475951 42 MARHY3613 _ Probable PspA protein +3.5 0.1716 0.6 FP475899 43 MARHY3634 _ Probable type VI secretion system subunit BH ND 0.2073 FP475884 44 MARHY3635 _ Probable type VI secretion system subunit +6.68 0.1118 0.7467 FP475883 45 MARHY3432 _ Probable TonB-dependent receptor +10.99 0.0193 0.2118 FP475902 46 MARHY3289 _ Probable porin PA 0.4185 ND FP475904 47 MARHY0478 _ Probable hydrophobic compounds transporter +11.23 0.0369 0.4144 FP475946 48 MARHY0477 _ Conserved protein +5.62 0.0369 0.2076 FP475947 49 MARHY0460 _ Conserved protein BH ND 0.696 FP475948 50 MARHY1073 _ Conserved protein PA 0.0385 ND FP475939 51 MARHY2963 _ Conserved protein +9.21 0.0326 0.3001 FP475914 52 MARHY3166 _ Conserved protein -14.81 3.612 0.2445 FP475908 53 MARHY3295 _ Conserved protein BH ND 0.0961 FP475903 54 MARHY0333 _ Conserved protein BH ND 0.1249 FP475950 55 MARHY3623 _ Probable type VI secretion system subunit +4.71 0.0142 0.0671 FP475895 56 MARHY3550 _ Conserved protein PA 0.1351 ND FP475900 57 MARHY2686 _ Conserved protein +22.01 0.0924 2.0331 FP475915 58 MARHY2432 _ Conserved protein PA 0.0642 ND FP475919
aPositive values represent overexpression in biofilm/hexadecane condition. Negative values represent underexpression in biofilm/hexadecane
condition. BH means that the protein is solely detected in biofilm/hexadecane condition. PA means that the protein is solely detected in
planktonic/acetate condition. bAverage percent of spot volume relative to total spot volume. ND, not detected.
25
Table 2
Phylogenetic distribution of MARHY0477 and MARHY0478 homologs.
Sequences similar to translated MARHY0477 and MARHY0478 were searched against the non-redundant GenBank CDS translations database,
using the BLAST program. aND not determined. bNumber of homologous genes and their percent peptide sequence identity in brackets. calk and
P450 indicate the presence of alkane hydroxylase and cytochrome P450 alkane monooxygenase genes respectively.
Strain Provenance Alkane
degradationa MARHY0477
homologsb MARHY0478
homologsb Alkane degradation
genesc
Marinobacter hydrocarbonoclasticus SP17 Marine sediment, Mediterranean Sea yes - - 2 alk, 2 P450
Marinobacter hydrocarbonoclasticus VT8 Oil well off the Vietnamese coast yes 2 (74%, 33%) 3 (98%, 74%, 74%) 3 alk, 1 P450
Marinobacter algicola DG893 Culture of a dinoflagellate yes 1 (59%) 1 (83%) 2 alk
Alcanivorax borkumensis SK2 Sea water, North Sea yes 2 (34%, 32%) 1 (40%) 2 alk, 1 P450
Alcanivorax sp. DG881 Culture of marine algae yes 6 (35%, 32%, 33%, 31%, 31%, 29%)
6 (54%, 54%, 50%, 48%, 46%, 42%)
2 alk, 2 P450
Bermanella marisrubri Sea water, Red Sea ND 1 (31%) 1 (45%) 1 alk
Moritella sp. PE36 Deep sea floor, Pacific Ocean ND 1 (29%) 1( 39%) 1 alk
26
Figures Fig. 1
27
Fig. 2
28
Fig. 3